The immune system uses a variety of mechanisms for attacking pathogens. However, not all of these mechanisms are necessarily activated after immunization. Protective immunity induced by immunization is dependent on the capacity of the vaccine to elicit the appropriate immune response to resist or eliminate the pathogen. Depending on the pathogen, this may require a cell-mediated and/or humoral immune response.
A substance that enhances the immune response when administered together with an immunogen or antigen is known as an adjuvant.
The Gram-negative bacterium Vibrio cholerae (V. cholerae) is the causative agent of the gastrointestinal disease cholera. The diarrhea caused by V. cholerae is due to the secretion of cholera toxin (CT).
CT comprises a single A subunit (CT-A), which is responsible for the enzymatic activity of the toxin, and five identical B subunits (CT-B), which are involved in the binding of the toxin to intestinal epithelial cells, as well as other cells which contain ganglioside GM1 on their surface. Together, the CT-A and CT-B subunits comprise a holotoxin. The sequence of CT has been described (Bibliography entry 1).
CT a is hexaheteromeric complex consisting of one A polypeptide and five identical B polypeptides (2). The B pentamer is required for binding to the cell surface receptor ganglioside GM1 (3). The A subunit can be proteolytically cleaved within the single disulfide-linked loop between C187 and C199 to produce the enzymatically active A1 polypeptide (4) and the smaller polypeptide A2, which links fragment A1 to the B pentamer (5). Upon entry into enterocytes, CT-A1 ADP-ribosylates a regulatory G-protein (Gsα), which leads to constitutive activation of adenylate cyclase, increased intracellular concentration of cAMP, and secretion of fluid and electrolytes into the lumen of the small intestine (6). In vitro, ADP-ribosyl transferase activity of CT is stimulated by the presence of accessory proteins called ARFs (7), small GTP-binding proteins known to be involved in vesicle trafficking within the eukaryotic cell.
The need for effective immunization procedures is particularly acute with respect to infectious organisms which cause acute infections at, or gain entrance to the body through, the gastrointestinal, pulmonary, nasopharyngeal or genitourinary surfaces. These areas are bathed in mucus, which contains immunoglobulins consisting largely of secretory IgA (8, 9, 10). This antibody is derived from large numbers of IgA-producing plasma cells which infiltrate the lamina propria regions underlying these mucosal membranes (11, 12). IgA is specifically transported to the lumenal surface through the action of the secretory component (13).
However, parenteral immunization regimens are usually ineffective in inducing secretory IgA responses. Secretory immunity is most often achieved through the direct immunization of mucosally-associated lymphoid tissues. Following their induction at one mucosal site, the precursors of IgA-producing plasma cells extravasate and disseminate to diverse mucosal tissues where final differentiation to high-rate IgA synthesis occurs (14, 15, 16). Extensive studies have demonstrated the feasibility of mucosal immunization to induce this common mucosal immune system (17), but with rare exceptions the large doses of antigen required to achieve effective immunization have made this approach impractical for purified vaccine antigens. Among the strategies investigated to overcome this problem is the use of mucosal adjuvants. It is known that CT is one of the most potent adjuvants, and that the co-administration of CT with an unrelated antigen results in the induction of concurrent circulating and mucosal antibody responses to that antigen (18). Thus, CT can act as an adjuvant.
It would be preferable to use as an adjuvant a form of the CT holotoxin that has reduced toxicity so as to reduce the undesirable symptoms of diarrhea caused by wild-type CT. Thus, there is a need to identify a mutant CT holotoxin which is able to enhance the immune response while reducing the toxicity of the CT holotoxin.